Arnulf Hartl

Non-anaphylactic surface-exposed peptides of the major birch pollen allergen, Bet v 1, for preventive vaccination

Background Almost 100 million allergic patients are sensitized to the major birch pollen allergen, Bet v 1, a 17 kDa protein containing most of the IgE epitopes present in pollens of trees belonging to the Fagales order and plant‐derived food.

Conditional Deletion of Histone Deacetylase 1 in T Cells Leads to Enhanced Airway Inflammation and Increased Th2 Cytokine Production

Chromatin modifications, such as reversible histone acetylation, play a key role in the regulation of T cell development and function. However, the role of individual histone deacetylases (HDACs) in T cells is less well understood. In this article, we show by conditional gene targeting that T cell-specific loss of HDAC1 led to an increased inflammatory response in an in vivo allergic airway inflammation model. Mice with HDAC1-deficient T cells displayed an increase in all critical parameters in this Th2-type asthma model, such as eosinophil recruitment into the lung, mucus hypersecretion, parenchymal lung inflammation, and enhanced airway resistance. This correlated with enhanced Th2 cytokine production in HDAC1-deficient T cells isolated from diseased mice. In vitro-polarized HDAC1-deficient Th2 cells showed a similar enhancement of IL-4 expression, which was evident already at day 3 of Th2 differentiation cultures and restricted to T cell subsets that underwent several rounds of cell divisions. HDAC1 was recruited to the Il4 gene locus in ex vivo isolated nonstimulated CD4+ T cells, indicating a direct control of the Il4 gene locus. Our data provide genetic evidence that HDAC1 is an essential HDAC that controls the magnitude of an inflammatory response by modulating cytokine expression in effector T cells.

Genetic engineering of allergens: future therapeutic products.

Genetic engineering of allergens for specific immunotherapy should aim at the production of modified molecules with reduced IgE-binding epitopes (hypoallergens), while preserving structural motifs necessary for T cell recognition (T cell epitopes) and for induction of IgG antibodies reactive with the natural allergen (blocking antibodies). Common approaches for engineering of hypoallergens usually require knowledge of T and B cell epitopes and involve changing specific base pairs (mutated gene), introduction of a new piece of DNA into the existing DNA molecule (chimeric or hybrid gene), and deletions (truncated gene or fragments). DNA family shuffling has the advantage that it does not require a priori knowledge of structural and functional properties for efficient generation of hypoallergens. The combination of the hypoallergen concept with the Th1-inducing genetic immunization approach might be an attractive alternative for protein-based immunotherapy.

Generation of an Allergy Vaccine by Disruption of the Three-Dimensional Structure of the Cross-Reactive Calcium-Binding Allergen, Phl p 7

The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost their structural fold. Allergic patients’ IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patients’ IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergen’s three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.

Costimulation Blockade Inhibits Allergic Sensitization but Does Not Affect Established Allergy in a Murine Model of Grass Pollen Allergy

Type I allergy is characterized by the development of an initial Th2-dependent allergen-specific IgE response, which is boosted upon a subsequent allergen encounter. Although the immediate symptoms of allergy are mainly IgE-mediated, allergen-specific T cell responses contribute to the late phase as well as to the chronic manifestations of allergy. This study investigates the potential of costimulation blockade with CTLA4Ig and an anti-CD154 mAb for modifying the allergic immune response to the major timothy grass pollen allergen Phl p 5 in a mouse model. BALB/c mice were treated with the costimulation blockers at the time of primary sensitization to the Phl p 5 allergen or at the time of a secondary allergen challenge. Costimulation blockade (CTLA4Ig plus anti-CD154 or anti-CD154 alone) at the time of sensitization prevented the development of allergen-specific IgE, IgM, IgG, and IgA responses compared with untreated but sensitized mice. However, costimulation blockade had no influence on established IgE responses in sensitized mice. Immediate-type reactions as analyzed by a rat basophil leukemia cell mediator release assay were only suppressed by early treatment but not by a costimulation blockade after sensitization. CTLA4Ig given alone failed to suppress both the primary and the secondary allergen-specific Ab responses. Allergen-specific T cell activation was suppressed in mice by early as well as by a late costimulation blockade, suggesting that IgE responses in sensitized mice are independent of T cell help. Our results indicate that T cell suppression alone without active immune regulation or a shifting of the Th2/Th1 balance is not sufficient for the treatment of established IgE responses in an allergy.

Prevention of allergen-specific IgE production and suppression of an established Th2-type response by immunization with DNA encoding hypoallergenic allergen derivatives of Bet v 1, the major birch-pollen allergen

In atopic patients, programming towards a preferential Th2 immunity leads to IgE antibody production and cellular Th2 immunity against otherwise harmless antigens. We report the development ofprophylactic and therapeutic DNA vaccines for the major birch‐pollen allergen, Bet v 1. We constructed three DNA vaccines, coding for the complete cDNA, coding for two hypoallergenic fragments or coding for a hypoallergenic Bet v 1 mutant. The protective effect was studied in mice pretreated by intradermal DNA injections, then sensitized with Bet v 1 protein. Mice pretreated with any of the three Bet v 1‐specific DNA vaccines were protected against allergic sensitization to Bet v 1. Protection was characterized by a lack of Bet v 1‐specific IgE production, a lack of basophil activation and an enhanced IFN‐γ expression. DNA vaccines with wild‐type Bet v 1 induced strong Bet v 1‐specific antibody responses whereas DNA vaccines with hypoallergenic Bet v 1 derivatives induced no (fragments) or only transient (mutant) Bet v 1‐specific antibody responses. A therapeutic approach with the fragment‐DNA vaccine reduced IgE production and stimulated a sustained Th1 cytokine milieu. Our results demonstrate that DNA vaccines with hypoallergenic forms of the allergen specifically protect against sensitization and suppress established Th2‐type responses. This concept may be applied for the development of safe and specific DNA vaccines for the prophylaxis and therapy of allergic diseases.

Isoforms of the Major Allergen of Birch Pollen Induce Different Immune Responses after Genetic Immunization

Background: Recent publications indicate that immunization with plasmid DNA encoding allergens might represent a potential approach in allergen–specific immunotherapy. Objective: In the present study we have compared the immune responses induced by plasmid DNA encoding for two isoforms of Bet v 1, the major allergen of birch pollen. Methods: BALB/c mice were injected intradermally with plasmid DNA encoding for the genes of Bet v 1a (pCMV–Beta) and Bet v 1d (pCMV–Betd). In addition, the effect of immunostimulatory DNA sequences was investigated by appending and/or coinjecting CpG motifs. Antibody responses and IFN–γ and IL–4 levels were measured by ELISA. Allergen–specific proliferation was determined by incorporation of [3H]–thymidine. Results: The two isoforms induced a similar humoral response. The lack of any IgE production and the ratio of IgG1 to IgG2a clearly indicated a Th–1–type response. The antisera against both isoforms were highly cross–reactive, which was supported by the energy plot indicating similar folding of the two protein isoforms. However, determination of IFN–γ and IL–4 in the serum elicited a strikingly different cytokine profile during the course of the immune response. In contrast to pCMV–Beta, pCMV–Betd caused no significant allergen–specific proliferation and induced only marginal levels of the key cytokines. Conclusions: Based on the assumption that the induction of a strong Th–1 type response is a prerequisite for successful treatment of allergy, our results favor the use of isoform Bet v 1a in combination with CpG motifs for a novel type of allergen immunotherapy based on plasmid DNA immunization. Additionally, the data also confirm the assumption that the antigen itself can have a marked influence on the immune response after genetic immunization.

Characterization of the protective and therapeutic efficiency of a DNA vaccine encoding the major birch pollen allergen Bet v 1a

Background:  An estimated 100 million individuals suffer from birch pollen allergy. More than 95% of birch pollen‐allergic subjects react with the major birch pollen allergen Bet v 1a, and almost 60% of them are sensitized exclusively to this allergen.

The Influence of CpG Motifs on a Protein or DNA-Based Th2-Type Immune Response against Major Pollen Allergens Bet v 1a, Phl p 2 and Escherichia coli-Derived β-Galactosidase

Background: DNA immunization and protein immunization with CpG motifs as adjuvants represent promising approaches in allergen-specific immunotherapy. Objective: We investigated the effect of coinjection or prepriming with CpG-ODN on Th2-type responses induced by gene gun and protein immunization. Methods: BALB/c mice were immunized with the gene gun using plasmid DNA containing the cDNAs coding for the genes of Bet v 1a, Phl p 2 and β-galactosidase or with the purified Al(OH)3-adsorbed proteins. In addition, CpG-ODN were applied by coinjection or by prepriming treatment. Antibody and cytokine responses were measured by ELISA, proliferative and cytotoxic responses were determined by standard labeling procedures. Furthermore, the allergenic activity of sera was measured by passive cutaneous anaphylaxis. Results: Gene gun immunization and protein immunization induced a clear Th2-type response for all antigens. The Th1-promoting effect of CpG-ODN coinjection together with gene gun immunization was restricted to β-galactosidase as indicated by the increase of IgG2a and a marked expression of IFN-γ. CpG motifs also increased the specific cytotoxic response against β-galactosidase. Prepriming with CpG-ODN and gene gun or protein immunization with Bet v 1a exhibited no significant difference to the non-CpG control group. However, sera from mice preprimed with CpG-ODN induced no anaphylaxis with gene gun immunization, but with protein immunization. Conclusions: The effect of CpG motifs in vivo depends on a variety of parameters like the nature of the antigen and the immunization modality. Furthermore, our studies indicate that a combination of CpG + DNA immunization may be more effective in antagonizing Th2 responses than the combination of CpG + protein immunization.

Low dose hypericin-PDT induces complete tumor regression in BALB/c mice bearing CT26 colon carcinoma.

BACKGROUND Successful tumor eradication with photodynamic therapy (PDT) in vivo depends on the optimal combination of treatment parameters. (Low-dose) PDT may additionally induce antitumoral immune responses. Since the naturally occurring hypericin (Hyp) is a promising photosensitizer for PDT, the aim of the study was to investigate phototoxic and immunologic effects of a low-dose Hyp-PDT on murine tumors in contrast to commonly used Hyp-PDT conditions. METHODS BALB/c mice bearing CT26 colon carcinoma received hypericin intravenously and were irradiated with red light 0.5-4h later. Tumor development was recorded. Mice were then re-challenged 60 days after the first tumor cell inoculation to investigate an antitumoral immune response. RESULTS Different results of tumor/host responses were obtained, ranging from mice exitus over delayed tumor growth to complete tumor regression according to different treatment protocols. PDT with common doses and a 4h drug-light-interval resulted in a four times delayed tumor growth compared to the control groups. PDT with relatively low doses and a drug-light-interval of 0.5h led to 100% tumor eradication. Re-challenge of these mice with CT26 mouse colon carcinoma cells prevented new tumor growth. CONCLUSIONS Not only drug concentrations and light doses seem to determine the efficiency of tumor eradication, but also the localization of hypericin at the time of irradiation. Targets in our low-dose PDT protocol are exclusively the vessels. The advantage of this low-dose PDT beside less drug and light exposure of the animals is reduced skin damage, faster healing of the lesions and induction of an antitumoral immune response.